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Trametes versicolor protein YZP activates regulatory B lymphocytes - gene identification through de novo assembly and function analysis in a murine acute colitis model.

Kuan YC, Wu YJ, Hung CL, Sheu F - PLoS ONE (2013)

Bottom Line: YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d.Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene.

View Article: PubMed Central - PubMed

Affiliation: Center for Biotechnology, National Taiwan University, Taipei, Taiwan ; Department of Horticulture, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized.

Results: We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.

Conclusions: We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d(+) Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models.

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YZP binds to B cells through TLR2 and TLR4 and activates the gene expression of TLR-signaling molecules.A. MACS-purified CD19+ B cells from WT, TLR2–/–, and TLR4–/– mice were pre-incubated with YZP (shaded grey), FITC-labeled YZP, or FITC-labeled BSA for 30 min. The cells were subsequently harvested for flow cytometry analysis. The numbers indicate the percentages of positive fluorescent cells among the total cells. The right panel shows the results from 3 independent experiments, presented as the mean ± SD (n = 3). The asterisks indicate significant differences (p<0.05) between TLR–/– and WT mice. B. MACS-purified CD19+ B cells were stimulated with YZP (10 μg/mL) for the indicated periods. Total RNA was extracted and subsequently reverse transcribed to cDNA for gene expression analysis. The asterisks indicate that the data are significantly different (p<0.05) from the 0 h control group.
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pone-0072422-g011: YZP binds to B cells through TLR2 and TLR4 and activates the gene expression of TLR-signaling molecules.A. MACS-purified CD19+ B cells from WT, TLR2–/–, and TLR4–/– mice were pre-incubated with YZP (shaded grey), FITC-labeled YZP, or FITC-labeled BSA for 30 min. The cells were subsequently harvested for flow cytometry analysis. The numbers indicate the percentages of positive fluorescent cells among the total cells. The right panel shows the results from 3 independent experiments, presented as the mean ± SD (n = 3). The asterisks indicate significant differences (p<0.05) between TLR–/– and WT mice. B. MACS-purified CD19+ B cells were stimulated with YZP (10 μg/mL) for the indicated periods. Total RNA was extracted and subsequently reverse transcribed to cDNA for gene expression analysis. The asterisks indicate that the data are significantly different (p<0.05) from the 0 h control group.

Mentions: The interaction between YZP and B cells was studied in TLR2 and TLR4 gene knockout mice with FITC-labeled YZP. Splenic B cells from WT, TLR2–/–, and TLR4–/– mice cultured with FITC-YZP or FITC-BSA were analyzed by flow cytometry. The populations of YZP+ cells in WT, TLR2–/–, and TLR4–/– B cells were 27%, 21%, and 11%, respectively. Compared with WT B cells, the YZP+ cell populations were reduced 40% and 65% in TLR2–/– and TLR4–/– B cells, respectively (Figure 11A). These findings are consistent with the reduced YZP activity observed in TLR2/4-blocked B cell cultures (Figure 9 and Figure 10). The expression of genes downstream of the TLR signaling pathway was studied by qPCR to determine if YZP activates B cells through TLR-mediated signals. Significant induction of MyD88, TIRAP, TRAF6, and NF-κB gene expression was observed within 3 h (Figure 11B), suggesting that the TLR signaling pathway is involved in the activation of B cells through YZP.


Trametes versicolor protein YZP activates regulatory B lymphocytes - gene identification through de novo assembly and function analysis in a murine acute colitis model.

Kuan YC, Wu YJ, Hung CL, Sheu F - PLoS ONE (2013)

YZP binds to B cells through TLR2 and TLR4 and activates the gene expression of TLR-signaling molecules.A. MACS-purified CD19+ B cells from WT, TLR2–/–, and TLR4–/– mice were pre-incubated with YZP (shaded grey), FITC-labeled YZP, or FITC-labeled BSA for 30 min. The cells were subsequently harvested for flow cytometry analysis. The numbers indicate the percentages of positive fluorescent cells among the total cells. The right panel shows the results from 3 independent experiments, presented as the mean ± SD (n = 3). The asterisks indicate significant differences (p<0.05) between TLR–/– and WT mice. B. MACS-purified CD19+ B cells were stimulated with YZP (10 μg/mL) for the indicated periods. Total RNA was extracted and subsequently reverse transcribed to cDNA for gene expression analysis. The asterisks indicate that the data are significantly different (p<0.05) from the 0 h control group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760908&req=5

pone-0072422-g011: YZP binds to B cells through TLR2 and TLR4 and activates the gene expression of TLR-signaling molecules.A. MACS-purified CD19+ B cells from WT, TLR2–/–, and TLR4–/– mice were pre-incubated with YZP (shaded grey), FITC-labeled YZP, or FITC-labeled BSA for 30 min. The cells were subsequently harvested for flow cytometry analysis. The numbers indicate the percentages of positive fluorescent cells among the total cells. The right panel shows the results from 3 independent experiments, presented as the mean ± SD (n = 3). The asterisks indicate significant differences (p<0.05) between TLR–/– and WT mice. B. MACS-purified CD19+ B cells were stimulated with YZP (10 μg/mL) for the indicated periods. Total RNA was extracted and subsequently reverse transcribed to cDNA for gene expression analysis. The asterisks indicate that the data are significantly different (p<0.05) from the 0 h control group.
Mentions: The interaction between YZP and B cells was studied in TLR2 and TLR4 gene knockout mice with FITC-labeled YZP. Splenic B cells from WT, TLR2–/–, and TLR4–/– mice cultured with FITC-YZP or FITC-BSA were analyzed by flow cytometry. The populations of YZP+ cells in WT, TLR2–/–, and TLR4–/– B cells were 27%, 21%, and 11%, respectively. Compared with WT B cells, the YZP+ cell populations were reduced 40% and 65% in TLR2–/– and TLR4–/– B cells, respectively (Figure 11A). These findings are consistent with the reduced YZP activity observed in TLR2/4-blocked B cell cultures (Figure 9 and Figure 10). The expression of genes downstream of the TLR signaling pathway was studied by qPCR to determine if YZP activates B cells through TLR-mediated signals. Significant induction of MyD88, TIRAP, TRAF6, and NF-κB gene expression was observed within 3 h (Figure 11B), suggesting that the TLR signaling pathway is involved in the activation of B cells through YZP.

Bottom Line: YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d.Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene.

View Article: PubMed Central - PubMed

Affiliation: Center for Biotechnology, National Taiwan University, Taipei, Taiwan ; Department of Horticulture, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized.

Results: We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.

Conclusions: We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d(+) Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models.

Show MeSH
Related in: MedlinePlus